Jocelyne Bach

Effects of the fungicide prochloraz on xenobiotic metabolism in rainbow trout: inhibition in vitro and time course of induction in vivo

1. Interactions between the fungicide prochloraz and the hepatic cytochrome P-450 of rainbow trout were studied by determination of enzymic activities in vitro using the microsomal fraction, and by kinetic studies. 2. Prochloraz inhibited 7-ethoxyresorufin-O-deethylase (EROD) in vitro. This inhibition was partially non-competitive: the enzyme-substrate-inhibitor (ESI) complex was catalytically active (68% of the activity of the enzyme-substrate complex). 3. Prochloraz in vitro inhibited aldrin epoxidase (AE) by a linear mixed-type mechanism. This inhibition might be high because of high affinity for prochloraz and lack of catalytic activity of the ESI complex. 4. Effects of prochloraz in vivo were studied as a function of time after intraperitoneal injection. Total cytochrome P-450 increased for more than 21 days. 5. EROD increased slightly at day 4 and then returned to control level. Kinetics showed an increase in apparent Km and Vmax. AE was strongly inhibited at day 4 (large increase in apparent Km) and then returned to control level in 21 days. 6. Spectral interactions with aniline showed strong inhibition and recovery to an activated level at day 21.

Ergosterol biosynthesis and its inhibition by fenpropimorph in Fusarium species

Abstract In the mycelium of all the Fusarium species studied, the major sterol was ergosterol in the absence of fenpropimorph. In the presence of this fungicide fenpropimorph-sensitive strains, with the exception of F. nivale , accumulated mainly Δ 8,14 -sterols and tolerant ones accumulated either both Δ 8,14 - and Δ 8 -sterols or only Δ 8 -sterols. This seemed to indicate that fenpropimorph toxicity was related to sterol Δ 14 -reductase sensitivity. In F. nivale , which was highly sensitive to fenpropimorph, the accumulation of only Δ 8 -sterols indicated that the main target enzyme was sterol Δ 8 →Δ 7 -isomerase. In addition to Δ 8,14 -and Δ 8 -sterols, squalene was accumulated in very high amounts in several strains; the level of squalene accumulation did not seem to be correlated with fenpropimorph sensitivity.

Effects of the fungicide prochloraz on xenobiotic metabolism in rainbow trout: In vivo induction

1. Rainbow trout were dosed with prochloraz by i.p. injection of sprayed food pellets. Cytochrome P-450, two P-450-dependent activities, and two conjugase activities were measured in vitro in microsomal or cytosolic fractions. 2. Prochloraz increased cytochrome P-450 in liver, intestine, and pyloric caeca: maximum response occurred at 30-100 mg/kg i.p. In cold conditions, this increase persisted for more than 8 days after injection. 3. Hepatic 7-ethoxycoumarin-O-dealkylase (ECOD) and 7-ethoxyresorufin-O-dealkylase (EROD) were inhibited by prochloraz except in one assay in warm water where they increased. In intestine and pyloric caeca, ECOD and EROD were not detected, even when cytochrome P-450 was increased. 4. UDP-glucuronosyltransferase (1-naphthol as substrate) was unchanged or inhibited after prochloraz dosing. 5. Glutathione-S-transferase (o-dinitrobenzene as substrate), was unchanged or inhibited by prochloraz. 6. The measured level of enzymic activities was the result of induction and inhibition by prochloraz residues. Variations in basal activities and perhaps in prochloraz interactions were due to temperature acclimatization.

Effects of prochloraz on drug metabolism in the Japanese quail, grey partridge, chicken, and pheasant.

Prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-1H-imidazole-1-carboxamide) is a new agricultural fungicide. When administered in the diet, it induced hepatic and intestinal cytochrome P-450 levels and monooxygenase activities from Japanese quail (Coturnix coturnix), grey partridge (Perdix perdix), chicken (Gallus gallus), and pheasant (Phasianus colchicus). After administration of a single dose to quail, prochloraz acted biphasically,i.e. inhibited, then induced aniline hydroxylase activity. In close relation with this biphasic pattern, pentobarbital sleeping time and toxicity of the insecticide parathion were modified. Administration of prochloraz at various dose levels in the diet of quail did not influence fecundity, fertility, and development of progeny.

Effects of sterol biosynthesis inhibitor fungicides in the phytopathogenic fungus, Nectria haematococca: ergosterol depletion versus precursor or abnormal sterol accumulation as the mechanism of fungitoxicity

The relative importance of the depletion of ergosterol versus the accumulation of precursor or abnormal sterols in the mechanism of fungal growth inhibition by sterol biosynthesis inhibitor fungicides is incompletely understood. In order to investigate this problem further, the degree of inhibition of the growth of Nectria haematococca by fungicides with different enzymatic targets in the sterol biosynthetic pathway was determined and compared with their effects on the sterol profile. The sensitivity of N. haematococca was highest towards fenpropimorph, followed by tebuconazole, terbinafine, fenpropidin and tridemorph. Terbinafine, a squalene epoxidase inhibitor, induced a very large accumulation of squalene without very significant inhibition of ergosterol biosynthesis and growth. The fungus appeared able to tolerate large amounts of squalene. In the case of tebuconazole, a sterol 14α-demethylase inhibitor, it seemed that the accumulation of C4 mono- and dimethyl sterols was responsible for fungitoxicity. Fenpropimorph and fenpropidin seemed to be good inhibitors of both sterol Δ14-reductase and Δ8Δ7-isomerase, whereas tridemorph was a better inhibitor of Δ8Δ7-isomerase than of the Δ14-reductase. Large accumulations of Δ8,14- or Δ8-sterols and correspondingly large decreases in the ergosterol content are both implicated in the fungitoxicity of these compounds in N. haematococca.

Hepatic biotransformation in the buzzard (Buteo buteo) and the Japanese quail (Coturnix coturnix): Effect of PCBs

Hepatic biotransformation was studied in microsomal (cytochrome P-450-dependent monooxygenase activities) and cytosolic (glutathione S-transferase activities) fractions from Japanese quail (Coturnix coturnix) and buzzard (Buteo buteo). Monooxygenase activities were not very different apart from a high 7-ethoxycoumarin de-ethylase activity in quail as compared to buzzard. Glutathione S-transferase activities were higher in quail than in buzzard. DP5 (a commercial mixture of PCBs containing 50% chlorine) produced a marked increase in monooxygenase activity from quail liver. In contrast, no induction was found in buzzard under the same conditions. Glutathione S-transferase activities were not modified in both species.

Role of sterol 3-ketoreductase sensitivity in susceptibility to the fungicide fenhexamid in Botrytis cinerea and other phytopathogenic fungi

BACKGROUND The narrow-spectrum fungicide fenhexamid was introduced into French vineyards in 2000 to control grey mould caused by a complex of two cryptic species: Botrytis cinerea, the predominant species sensitive to fenhexamid, and Botrytis pseudocinerea, naturally resistant. Fenhexamid was suggested to inhibit the 3-ketoreductase involved at C-4 demethylation steps during ergosterol biosynthesis, as revealed by its effects on the B. cinerea sterol profile. Resistance monitoring studies have hitherto identified two B. cinerea fenhexamid-resistant phenotypes, both resulting from mutations in the erg27 gene encoding 3-ketoreductase. RESULTS The role of 3-ketoreductase sensitivity in fungal susceptibility to fenhexamid was investigated by studying sterol profiles and microsomal 3-ketoreductase in various fungal strains. Fenhexamid does inhibit B. cinerea 3-ketoreductase activity. Erg27 mutations causing amino acid substitutions in or near the transmembrane domain strongly decrease the affinity of fenhexamid for 3-ketoreductase. Fenhexamid has very low affinities for 3-ketoreductase in inherently resistant species, whether closely related to B. cinerea, like B. pseudocinerea, or more distantly related, like Nectria haematococca. CONCLUSION erg27 mutation and erg27 polymorphism may therefore contribute to the unfavourable binding of fenhexamid to its target, 3-ketoreductase, explaining the acquisition of fenhexamid resistance in B. cinerea and the narrow spectrum of this fungicide.

The effects of temperature on the basal activity of cytochrome P-450 in rainbow trout (Salmo gairdneri)

1. 1. In trout acclimatized for 8 days to various temperatures, cytochrome P-450 and some related activities were measured in liver microsomes. 2. 2. The protein and cytochrome P-450 contents were unchanged with temperature. 3. 3. Aldrin epoxidation did not show compensation with temperature. 4. 4. 7-Ethoxyresorufin-O-deethylase activity showed a large increase at 11°C with a low Km and a high max. 5. 5. 7-Ethoxycoumarin-O-deethylase showed maximum activity at 11°C. 6. 6. The levels of cytochrome P-450-mediated activities varied differently with temperature as a function of the various isozymes involved.

Effects of chloramphenicol on hepatic cytochrome P-450 in rainbow trout

Abstract 1. In microsomes from trout liver, chloramphenicol inhibited 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro according to a two-step mechanism: competition with substrate followed by irreversible fixation. 2. Chloramphenicol inhibited aldrin epoxidase (AE) activity in vitro only under a concentration higher than a given threshold. 3. In vivo , the response of AE activity to increasing doses of chloramphenicol was similar. 4. Two steps were observed after dosing trout with chloramphenicol: firstly, an AE and EROD inhibition which was conceivably only due to residual chloramphenicol in liver and lasted for less than 8 days; then, AE and EROD were activated (increase of V max , but with high K m ) up to 21 days after dosing.